Many diseases in humans are associated with diminished general cellular immune response or anergy. This defect is exhibited most prominently in congenital immunodeficiency diseases but also occurs in many patients with viral, bacterial, or fungal infections, as well as in so-called autoimmune diseases and cancer. Cellular immunodeficiency or anergy may also be induced by immunosuppressive therapy in the form of drug or radiation therapy. Regardless of the cause, the effect of this deficiency on the patient is a reduced ability to generate an adequate cellular immune response to invading organisms or "foreign" cells or tissues.
The most frequently used in vivo method for assessing cellular immunity in humans is delayed hypersensitivity skin testing. In this skin test, minute quantities of antigens are injected into the skin of the patient (intradermally), and the resultant reaction is measured at designated intervals after injection.
This delayed hypersensitivity skin reaction has a series of in vitro correlates which are understood to represent components or integral parts of the skin reaction. The in vitro tests measure the activity of one or more biological factors or kinins, also known as lymphokinins, and elicit responses from patient blood cells using the same antigens that are used in skin testing.
For determination of general cellular immunocompetence, the antigens used are so-called "recall" antigens, which are bacterial or viral extracts the use of which in the above-described in vivo and in vitro tests stems from the ability of normal individuals to remember or "recall" a cellular immune response due to sensitization early in life. Antigens most commonly used are tuberculin antigens (such as Purified Protein Derivative-Tuberculin (PPD)), Streptokinase-Streptodornase (SK-SD), Candida Albicans, Mumps, Tetanus Toxoid, Trichophyton, Histoplasmin, and Coccidiodin, but any other art-recognized recall antigen may be used.
With regard to tuberculin antigens in particular, it should be noted that there are a great number of these which have been extracted from or produced by the tubercle bacillus, including PPD, bacillus calmette Guerin (BCG), New tuberculin, Mantoux tuberculin, and the like. See, for example, the definition of "tuberculin" in Dorland's Medical Dictionary, 25th edition, W. B. Saunders, Philadelphia, 1974, for a list of common tuberculin antigen preparations.
At present, skin testing and in vitro assays employing "recall" antigens are used: (1) to determine anergy in selected patients, (2) to evaluate the results of immunotherapy for cancer and other diseases that have immunodeficiency as a major component, as either a cause or a result of the disease, (3) to monitor the severity of induced immunosuppression or (4) to follow the course of a disease process. It is well known that drug or radiation-induced suppression is an unavoidable byproduct of the treatment for many forms of cancer and the maintenance of organ transplant recipients. See, for example, L. E. Spitler, "Delayed Hypersensitivity Skin Testing" in Manual of Clinical Immunology, N. R. Rose and H. Friedman, et al., American Society of Microbiology, 1976.
Moreover, many tumors are characterized by specific immune responsiveness to the tumor antigens, which immune responsiveness is presently measured by a delayed hypersensitivity skin test as described above but with a tumor antigen substituted for the recall antigen. For many tumors, a correlation has been established between the degree of tumor specific immune response and the clinical state of the patient. A cellular immune response to a tumor-specific antigen in a tumor-bearing patient generally indicates a favorable clinical prognosis or outcome. After treatment of a patient by a variety of therapeutic techniques, a positive immune response in a patient who lacked such a response prior to treatment may be interpreted as a favorable prognosis and is correlated with disease remission. Conversely, a negative immune response in a patient who demonstrated a satisfactory response prior to treatment may be interpreted as an unfavorable prognosis and is not correlated with disease remission. Such skin tests may be used as evidence of an immune response to a tumor or tumor antigen, and may be used in conjunction with skin test employing "recall" antigens. See, for example, F. K. Nkrumah, et al., Int. J. Cancer: 20, 6-11 ( 1977) and D. H. Char, et al., N.E.J. Med: 291 (6), 274-277 (1974).
The prior art skin testing technique is well known to yield variable results due to differences in the dose of injected antigen, improper deposition of the antigen in the skin, instability of the antigen employed, and subjectivity involved in the reading of the reaction.
Moreover, skin testing is invasive and thus risks acute localized and systemic reactions in some individuals. This fact also makes skin testing unsuitable for repeated uses (e.g., monitoring) because repeated injections of antigen artificially boost the patient's immune response.
Finally, skin test procedures are not only associated with discomfort for the patient but also require reexamination of the patient 24 and 48 hours after testing to read the results. This necessity for observing the results is inconvenient, especially in a hospital setting (where weekends may intervene), which makes skin testing uncommonly used among outpatients.
Because skin testing has been known to involve these disadvantages and medical risks, recent efforts have focused on the use of the in vitro correlates for the above diagnostic purposes. The in vitro tests are noninvasive and do not risk acute systemic reactions (as are sometimes encountered with skin testing). In addition, these in vitro methods do not boost or artificially amplify the patient's immune response, as does the repeated use of skin tests.
Thus, only the in vitro methods are suitable for repeat testing that is needed to monitor patients who require potentially immunosuppressive therapy over time. These methods are less costly and safer than the skin test method. Unfortunately, however, no practical, portable in vitro test has been available in the past.
The present invention is based on the principle of Leukocyte Migration Inhibition (LMI). The basic elements of this cellular reaction were previously known. Lymphocytes obtained from patients who have been previously exposed (sensitized) to an antigen, upon reexposure to that antigen, elicit a defined protein factor referred to as Leukocyte Migration Inhibition Factor (LIF). This factor, which is one of a group of factors produced under the stated condition, causes the granulocytes from the same patient to be blocked or inhibited in movement (migration) in a variety of fluids or media. Inhibition of migration is interpreted as a positive, immunocompetent response to a given antigen. By using a variety of antigens, a profile is obtained to express the immune status of the patient at the time of testing.
Much of the previously known work on the LMI method is attributed to Clausen (Danish Med. Bull. 22(5):181-194; Acta Allergol. 28:145-158, 1973). He demonstrated that Leukocyte Migration Inhibition Factor (LIF) specifically causes inhibition of migration of granulocytes, can be measured in a direct, one stage test in agarose using peripheral blood lymphocyte-granulocyte mixtures or as a two-stage procedure in which cell free supernatant fluids (containing LIF) from antigen-stimulated lymphocyte cultures are assayed for migration inhibitory activity when added to purified leukocytes or granulocytes. Others have defined a relationship between the LMI method and delayed hypersensitivity skin testing (Astor and Fudenberg, J. Immunol. 110(4):1174, 1973).
The prior art LMI assay method requires the preincubation of a "recall" antigen at various concentrations with the leukocyte suspension from the patient and thus requires the fresh preparation of antigen and a mixing and incubation step prior to the test itself. Because of these features, the prior art LMI assay method requires specialized equipment and incubation of a cell suspension and hence is only slightly used. Specialized equipment often used in the prior art method includes, for example, a CO.sub.2 -perfused, temperature-controlled, water-jacketed 37.degree. C. incubator and a binocular microscope equipped with a micrometer eyepiece. The pre-incubation requirement has prevented development of a convenient, portable test of broad general application.
The novel LMI method described herein uses individual antigens premixed in agarose slides or plates, at a single predetermined antigen concentration, requiring no antigen-cell preincubation to activate the lymphoid cells. Such a system is more rapid and reproducible, encourages standardization of the methodology, and has heretofore been unavailable.
That the subject LMI method works at all is most surprising in view of the results reported by Clausen in the above-described Acta Allergologica article. He attempted to perform an LMI assay in which the recall antigen (PPD) was mixed with the agarose used to prepare the assay plates, rather than being pre-incubated with the leukocytes. The reported results (beginning at the bottom of page 67 of the article) caused Clausen to conclude:
"Leukocytes from tuberculin-positive persons without the addition of PPD, cultured in agarose medium containing PPD showed none, or only slight tuberculin induced migration inhibition, but if the same leukocytes were preincubated with PPD and placed in a medium that did not contain PPD, the migration inhibition was seen clearly." (ibid, p. 75; emphasis added).
The described invention utilizes plates or slides containing agarose with a "recall" or tumor specific antigen incorporated into the agarose of each plate at a defined diagnostically effective concentration. Patient leukocytes are added directly to wells within the agarose, requiring no preincubation between antigens and cell suspensions prior to this addition.